CN117737029B

CN117737029B - Glycosyltransferase mutant and application thereof in synthesis of collaterals plug

Info

Publication number: CN117737029B
Application number: CN202311800379.XA
Authority: CN
Inventors: 李国四; 易善勇; 王燕蝶; 王威; 韩邦兴
Original assignee: West Anhui University
Current assignee: West Anhui University
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-06-07
Anticipated expiration: 2043-12-25
Also published as: CN117737029A

Abstract

The invention discloses a glycosyltransferase mutant and application thereof in the synthesis of a complex plug, wherein the mutant is one of the following: mutating phenylalanine at position 111 of the amino acid sequence shown in SEQ ID NO.1 into tryptophan; phenylalanine at position 175 is mutated to aspartic acid. According to the invention, through a molecular transformation method combining directed evolution and semi-rational design, the glycosylation activity of the glycosyltransferase derived from Bacillus licheniformis on cinnamyl alcohol is obviously improved, and compared with the wild type mutant, the specific enzyme activity is respectively improved by 2.9 times and 2.5 times. The titer of the synthesized plug of cinnamyl alcohol catalyzed by the mutant can reach 7.1g/L, the conversion rate is 96.0%, and the space-time yield is 295.8mg/L/h. The invention solves the problem of low glycosyltransferase activity in the process of preparing the plug by an enzyme method, and has important industrial application prospect.

Description

Glycosyltransferase mutant and application thereof in synthesis of collaterals plug

Technical Field

The invention relates to a glycosyltransferase mutant and application thereof in the synthesis of a complex plug, belonging to the fields of biosynthesis and enzyme engineering.

Background

Rhodiola rosea is a traditional herbal medicine and has the physiological activities of resisting aging, resisting fatigue, enhancing immunity, protecting cardiovascular and cerebrovascular diseases and resisting tumor. Since the 60 s of the 20 th century, scientists have conducted extensive research on chemical active ingredients and pharmacological actions of rhodiola rosea as an environmentally-adaptive emerging herbal medicine. The analysis shows that the main active components of the composition comprise the collaterals plug, the collaterals plug and the derivatives of the collaterals plug and the collaterals plug. The collaterals plug and its derivative have antioxidant, physical endurance improving, psychological stress relieving, and memory improving effects. The chemical name of the cinnamyl monoglycoside is (2R, 3S,4S,5R, 6R) -2- (hydroxyymethyl) -6- [ (E) -3-phenylprop-2-enoxy ] oxane-3,4,5-triol or Trans-cinnamyl-O-beta-D-glucopyranoside, the molecular formula is C ₁₅H₂₀O₆, the molecular weight is 296.32, the CAS number is 85026-55-7, and the structural formula is as follows

Currently, the synthetic modes of the collaterals plug include several modes of plant extraction, chemical synthesis, plant cell transformation and microbial cell synthesis. Because of the over-exploitation, the wild resources of Rhodiola rosea have been increasingly scarce, rhodiola rosea has been listed as an endangered species in many countries, and the coronets and their derivatives coronets and coronets are present in only a few Rhodiola species, such as Rhodiola sachalinensis (Rhodiola sachalinnsis), rhodiola himalaica (Rhodiola himalensis), rhodiola rosea (Rhodiola rosea), rhodiola dentalis (Rhodiola serrata) and the like, in contents of usually only 0.1% to 3%, and therefore the method of extracting coronets from plants has become increasingly infeasible, development of a new preparation process of the plug is required. Currently, the main methods for chemically synthesizing glycoside compounds include the following: (1) Koenigs-Knorr glycosylation reaction: the glycoside is prepared by reacting an alpha-halo sugar with an alcohol under the catalysis of silver carbonate. This is the most common synthetic method, but requires the synthesis of glycosyl halides and expensive silver reagents; (2) Schmidt trichloroimidization reaction: trichloroacetonitrile is used for adding with glycosyl hemiacetal under alkaline condition to generate trichloroacetyl imine ester, and then the trichloroacetyl imine ester is reacted with alcohol or phenol through Lewis acid catalysis to prepare glycoside. However, this method involves the use of the carcinogen trichloroacetonitrile and the production of the genotoxic by-product trichloroacetamide; (3) Kahne glycosylation reaction: the glycosyl sulfoxide is used for preparing the glycoside compound through activation of trifluoromethanesulfonic anhydride and reaction with alcohol or phenol. The method requires lower temperature (-30 ℃ to-78 ℃) conditions, and is more severe; (4) other glycoside Synthesis methods: mainly, some methods, such as a phase transfer catalytic method and a trifluoroacetate method, are improved on the above methods, but they have some problems or limitations. The plug can also be obtained by means of plant cell transformation. For example, furmanowa et al used a callus culture method of rhodiola rosea, added cinnamyl alcohol to a suspension cell culture, researchers such as successfully obtained the conifer and its derivative conifer vitamin (Furmanowa M,Oledzka H,Michalska M,Sokolnicka I,Radomska D(1995)"Rhodiola rosea L.(Roseroot):in vitro regeneration and the biological activity of roots."In:Bajaj YPS(ed)Biotechnology in agriculture and forestry,vol 33.Medicinal and Aromatic Plants VIII(pp 412–426).Springer,Berlin).Grech-Baran used a rooting culture method, and successfully obtained the conifer and its derivative conifer vitamin (Grech-Baran M,Baranek K,Krajewska-Patan A,Pietrosiuk A(2014)"Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild-type and hairy root cultures of Rhodiola kirilowii."Biotechnol Lett 36:649–656). plant cell transformation by adding cinnamyl alcohol to the culture medium, which is milder and more environment-friendly than chemical synthesis in the reaction process, but lower yield limited the further development and utilization of this mode. From the viewpoint of synthetic biology, constructing a microbial heterologous biosynthesis pathway is a novel way for preparing a plug. Liu Tao et al constructed heterologous biosynthesis pathway of the plug in E.coli, and produced the plug with up to 280mg/L by fermenting glucose with recombinant E.coli (Chinese patent CN 108203713B). The method has low conversion rate, troublesome product separation and purification and further improves the yield.

Compared with the method, the process for preparing the plug by converting the cinnamyl alcohol in an enzyme catalysis mode has the advantages of short steps, single product configuration, high selectivity, high yield, simple production operation, low equipment requirement, environmental protection and great development prospect. Glycosyltransferases (glycosyltransferases, GT; EC 2.4. X.y) are a class of enzymes capable of catalyzing the transfer of a glycosyl moiety from an activated glycosyl donor molecule to a specific glycosyl acceptor molecule and the formation of a glycosidic bond. Catalytic glycosylation is a key step for synthesizing glycoside compounds with complex and diverse structures and physiological activity. Compared with a chemical method, the glycosylation reaction mediated by glycosyltransferase does not need complicated protection and deprotection, does not need to use catalysts with harmful effects on human bodies and environment, such as red phosphorus, bromine and the like, and is an environment-friendly biological modification method. Therefore, glycosylation of cinnamyl alcohol synthetic tampons by glycosyltransferases is of increasing interest. Since most natural products with important pharmacological effects are found in plants, plant-derived glycosyltransferases are the main research object of people, however, plant-derived glycosyltransferases have the problems of difficult expression and serious inclusion bodies, and limit the application of glycosyltransferases in the synthesis of collaterals. Compared with plant-derived glycosyltransferase, the microbial glycosyltransferase is easy to adopt the mode strains such as Saccharomyces cerevisiae, escherichia coli, bacillus subtilis and the like for heterologous expression, and is easier to realize industrial production. However, the reported microbial source glycosyltransferase catalyzed glycosylation activity is too low in the face of the unnatural substrate molecule cinnamyl alcohol, which not only reduces the yield of the plug but also greatly increases the catalyst cost. Therefore, the improvement of catalytic activity of microbial glycosyltransferase on cinnamyl alcohol through molecular transformation is a key for realizing the synthesis of the luosai enzyme method.

Disclosure of Invention

Aiming at the problem of low activity of microbial glycosyltransferase, the invention obtains the glycosyltransferase mutant with high selectivity to the alcoholic hydroxyl group of cinnamyl alcohol by carrying out site-directed saturation mutation on amino acid at a substrate binding pocket, thereby remarkably improving the yield of the plug.

In order to solve the technical problem of low glycosyltransferase activity, the first object of the invention is to provide a glycosyltransferase mutant, which comprises the following specific components:

Mutating phenylalanine at position 111 of the amino acid sequence shown in SEQ ID NO.1 into tryptophan; or (b)

Phenylalanine at position 175 of SEQ ID NO.1 was mutated to aspartic acid.

The second object of the present invention is to provide a nucleic acid molecule encoding the aforementioned glycosyltransferase mutant.

The third object of the present invention is to provide an expression vector carrying the aforementioned nucleic acid molecule.

The fourth object of the present invention is to provide a microorganism expressing the above glycosyltransferase mutant, or carrying the above nucleic acid molecule or the above expression vector.

As a specific example, the microorganism includes, but is not limited to, genetically engineered bacteria.

The fifth object of the present invention is to provide the use of the above glycosyltransferase mutant, or the above nucleic acid molecule, or the above expression vector, or the above microorganism in the synthesis of a plug.

As a specific example, the synthesis process of the plug is as follows:

under the existence of UDP-glucose provided by a UDP-glucose circulating system, the substrate cinnamyl alcohol is subjected to glycosylation reaction under the catalysis of a glycosyltransferase mutant to generate a plug. Specifically, firstly preparing enzyme solution of a glycosyltransferase mutant, adding the enzyme solution or genetically engineered bacteria capable of expressing the glycosyltransferase mutant into a substrate cinnamyl alcohol, mixing with a UDP-glucose circulating system, and carrying out glycosylation reaction in the mixed system to prepare the obtained plug.

Wherein the reaction temperature is 15-50 ℃, the pH value of the reaction system is 6-10, the concentration of UDP-glucose is 0.1-2 mM, and the concentration of DMSO is 1-5%.

The enzyme solution can be crude enzyme solution of cell disruption of genetically engineered bacteria expressing the glycosyltransferase mutant or purified enzyme solution.

The specific cycle process of the UDP-glucose circulating system is as follows: in the presence of UDP, sucrose is catalyzed by sucrose synthase to generate glucose, fructose and UDP-glucose, and the generated UDP-glucose participates in the synthesis of the collaterals plug again. Sucrose concentration is 50-600 mM.

The invention achieves the beneficial technical effects that: the glycosyltransferase adopted by the invention is a microbial source, and compared with the glycosyltransferase of plant sources, the glycosyltransferase of the invention is easier to be efficiently and soluble expressed in mode strains such as escherichia coli and the like; the glycosylation activity of the glycosyltransferase mutant obtained by the invention on the cinnamyl alcohol is obviously improved, 0.67U/mg is improved to 1.95U/mg and 1.63U/mg, and the glycosylation activity of the F111W and F175D mutants is 2.9 times and 2.5 times of that of the wild glycosyltransferase respectively; the titer of the synthesized plug of cinnamyl alcohol catalyzed by the mutant can reach 7.1g/L, the conversion rate is 96.0%, and the space-time yield is 295.8mg/L/h. The invention successfully solves the key problem of low glycosyltransferase activity in the process of preparing the complex plug enzyme method, and the obtained mutant has a very large industrial application prospect.

Drawings

FIG. 1 shows the effect of SDS-PAGE analysis on the overexpression of glycosyltransferases in E.coli; wherein M is marker, S is induced crushing supernatant, and P is induced crushing precipitate;

FIG. 2 is a diagram of key amino acid residues near the substrate pocket of glycosyltransferase BlYjiC;

FIG. 3 is a liquid chromatogram of a wild-type glycosyltransferase and glycosyltransferase mutant catalyzed;

FIG. 4 shows specific enzyme activities of wild-type glycosyltransferase WT-BlYjiC and mutants;

FIG. 5 is a schematic diagram of a catalytic preparation of a plug by a glycosyltransferase mutant;

FIG. 6 is a graph showing the time profile of wild type glycosyltransferase and its mutant in catalyzing cinnamyl alcohol to form a plug;

FIG. 7 is a high resolution mass spectrum of a plug prepared by glycosyltransferase mutant F111W;

FIG. 8 is a nuclear magnetic resonance ¹ H pattern (400 MHz) of a plug prepared by glycosyltransferase mutant F111W;

FIG. 9 is a nuclear magnetic resonance ¹³ C spectrum (400 MHz) of a plug prepared by glycosyltransferase mutant F111W catalysis.

Detailed Description

The invention is further illustrated in the following drawings and examples of embodiments, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The experimental methods are conventional methods unless specified, and the gene cloning operation can be specifically described in the molecular cloning experimental guidelines of J.

The DNA polymerase (2X Phanta Max Master Mix), dpn I enzyme, recombinant cloning kit and plasmid extraction kit used in the examples of the present invention were all purchased from Takara Biotechnology Co., ltd; the synthesis of genes and primers and the gene sequencing work are completed by general biology (Anhui) Inc., and the above reagent application method is referred to in the commodity instruction book.

The expression vector of the invention is pET-28a (+), and the host used is escherichia coli BL21 (DE 3), which are all preserved in the laboratory of the inventor.

All cinnamyl alcohol, UDP and UDP-glucose used in the invention are purchased from Shanghai Michelin Biochemical technologies Co., ltd; other commonly used reagents are available from Annaiji chemical Co.

The three-letter or one-letter expression of amino acids used in the present invention uses the amino acid codes specified by IUPAC (eur.j. Biochem.,138:9-37,1984).

EXAMPLE 1 cloning of glycosyltransferase

The wild type glycosyltransferase gene, the encoded amino acid sequence of which is shown in Table 1 and designated as WT-BlYjiC, was amplified using the Bacillus licheniformis (Bacillus licheniformis) ATCC14580 genome as a template and F and R as primers, and the amplified WT-BlYjiC gene was ligated to the pET-28a (+) plasmid (C-terminal His-tag purification tag) by means of a one-step cloning kit (ClonExpress II One Step Cloning Kit) to construct recombinant plasmid pET28a-BlYjiC. And transforming the recombinant plasmid into E.coli BL21 (DE 3) competent cells, screening positive clones, and detecting to obtain recombinant engineering bacteria containing the WT-BlYjiC gene.

TABLE 1 cloning of primers for glycosyltransferases

Primer name	Primer sequences	SEQ ID NO.
			F	aagaaggagatatactgcagATGGGACATAAACATATCGCGA	4
R	cggagctcgaattcggatccTTATTTTACTCCTGCGGGTGC	5

Note that: the lower case base is a recombination connection part with the carrier

SEQ ID NO.1 WT-BlYJiC

MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFMAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

EXAMPLE 2 expression and purification of wild-type glycosyltransferase WT-BlYjiC

The recombinant engineering bacteria strain was inoculated into 5mL LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured on an orbital culture shaker at 37℃for 12 hours at a speed of 200 rpm. 1mL of the culture was placed in a 250-mL flask containing 50mL of LB medium, and aerobic culture was performed at 37℃and 200 rpm. When OD ₆₀₀ reached about 0.6-0.8, cells were induced with 0.1mM IPTG. Cells were harvested after induction at 18 ℃ for 24 hours. Cells were centrifuged, resuspended in 50 mm potassium phosphate buffer pH 8.0 and disrupted by sonication. The cell extract was centrifuged at 12000rpm for 20 minutes, the supernatant was a crude enzyme solution, and SDS-PAGE analysis was performed on the supernatant and the pellet, respectively, to detect the expression of WT-BlYjiC, and the result is shown in FIG. 1. The target protein is found in the supernatant, and the WT-BlYjiC can be expressed in escherichia coli in a high-efficiency and soluble way, so that the expression effect is better than that of most plant-derived glycosyltransferases.

The wild type glycosyltransferase WT-BlYjiC was purified by nickel column affinity chromatography:

1) Sample pretreatment: re-suspending the cells with a loading buffer (20 mM phosphate buffer, pH 8.0, 25mM imidazole, 500mM NaCl), sonicating in an ice bath, centrifuging at 12000rpm to remove cell debris, and filtering the supernatant with a 0.22 μm filter;

2) Loading: washing the nickel column with 10 times of column volume loading buffer solution, and slowly loading the pretreated sample in the step 1) onto the column to fully combine the protein and the nickel column;

3) Washing: the nickel column was washed with 10 column volumes of wash buffer (20 mM pH 8.0 phosphate buffer, 50mM imidazole, 500mM NaCl);

4) Eluting target protein: eluting the target protein with elution buffer (20 mM pH 8.0 phosphate buffer, 250mM imidazole, 500mM NaCl);

5) Ultrafiltration and preservation: transferring the collected eluent into an ultrafiltration tube, and desalting and concentrating a protein sample through centrifugal ultrafiltration at 4 ℃; adding glycerol with final concentration of 20% into the ultrafiltered protein sample, quick-freezing with liquid nitrogen, and storing at-80deg.C.

Example 3 Activity assay of wild-type glycosyltransferase WT-BlYjiC

50MM potassium phosphate buffer (pH 8.0), 2mM cinnamyl alcohol, 5mM UDP-glucose, and an appropriate amount of enzyme were mixed, and shaken at 30℃and 1000 rpm. The reaction was quenched by the addition of 4 volumes of methanol and analyzed by High Performance Liquid Chromatography (HPLC). The unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μm of product per minute at standard assay conditions, 1 activity unit. HPLC detection was performed using a C18 column (ZORBAX SB-C18, agilent, USA) and the glycosylated product was analyzed at 30℃at a flow rate of 1mL/min at a detection wavelength of 275nm.

EXAMPLE 4 site-directed saturation mutagenesis of wild-type glycosyltransferase WT-BlYjiC

As shown in FIG. 2, the present invention screens out amino acids near the substrate binding pocket, including E41, E42, I62, D63, I67, L70, M77, F111, M112, Q136, F140, M174, F175, M320, P321 and I325, as subsequent site-directed saturation mutation sites by homology modeling.

According to the invention, a specific primer (shown in Table 2, SEQ ID NO.6-37 is sequentially arranged from top to bottom) is designed, mutation is introduced into a wild glycosyltransferase WT-BlYjiC gene sequence by using a QuickChange technology (An efficient onestepsite-directed and site-saturation mutagenesis protocol[J].Nucleic AcidsResearch,2004,32(14):e115), and a 41,42,62,63,67,70,77,111,112,136,140,174,175,320,321 and 325 site saturated mutation library is constructed.

Specifically, the PCR reaction system is as follows: ddH ₂ O2. Mu.L, DNA polymerase 5. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, template 1. Mu.L. PCR amplification conditions: 1) Pre-denaturation at 94℃for 2min; 2) Denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 40s (30 cycles of this step); and then extending at 72 ℃ for 5min. The PCR product was digested with Dpn I restriction enzyme at 37℃for 20 min. The digested product was then transformed into E.coli BL21 (DE 3) competent cells, plated on LB/Kan solid plates and incubated overnight at 37 ℃.

TABLE 2 rational design of primers for wild-type glycosyltransferase WT-BlYjiC

Note that: the bolded part is the post-mutation degenerate codon

EXAMPLE 5 mutant library screening

The mutant plate at each site was picked 96 clones and transferred to 96 deep well plates containing 50. Mu.g/mL kanamycin and 500. Mu.L LB liquid medium for culture, and when OD ₆₀₀ reached 0.6-0.8, cells were induced with 0.1mM IPTG. Centrifuging to remove supernatant, repeatedly freezing and thawing for 3 times, and using the supernatant as crude enzyme for catalytic reaction. The reaction system contained 2mM cinnamyl alcohol, 5mM UDP-glucose, 1% DMSO (1% by volume) and 50. Mu.L of 50mM potassium phosphate buffer (pH 8.0) of crude enzyme solution. The reaction was incubated at 30℃and 200rpm for 15min and terminated with 4 methanol volumes. The detection method described in example 3 is used for detecting the vitality and carrying out gene sequencing on the mutant with obviously improved vitality, wherein the liquid chromatogram after the catalysis of the wild type glycosyltransferase and the glycosyltransferase mutant is shown in figure 3, and the yield of the complex plug synthesized by the catalysis of the two glycosyltransferase mutants is obviously improved compared with that of the wild type glycosyltransferase. The results showed that F111W and F175D obtained a significant improvement over wild-type viability. The BlYJiC-F111W amino acid sequence is shown as SEQ ID NO.2, and the BlYJiC-F175D amino acid sequence is shown as SEQ ID NO.3. Glycosyltransferases and their mutants were purified as described in example 2 and their specific enzyme activities were determined as described in example 3 and the results are shown in FIG. 4. The results show that the specific activities of the mutants F111W and F175D are respectively improved from wild type 0.67U/mg to 1.95U/mg (F111W) and 1.63U/mg (F175D) which are respectively 2.9 and 2.5 times that of the wild type, and further show that the activities of the mutants F111W and F175D are obviously improved. SEQ ID NO.2: blYJiC-F111W

MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDWMAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

SEQ ID NO.3:BlYJiC-F175D

MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFMAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMDEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

EXAMPLE 6 glycosyltransferase and mutant thereof for preparing a Corp

The preparation of the complex plug adopts a crude enzyme double enzyme coupling system to carry out glycosylation of cinnamyl alcohol and in-situ regeneration of UDP-glucose, the preparation process is shown in figure 5, and sucrose synthase catalyzes sucrose to generate glucose, fructose and UDP-glucose in the presence of UDP, so that UDP-glucose generated by the reaction can participate in the complex plug synthesis again, and the continuous progress of the reaction is ensured. Wherein the sucrose synthase is derived from soybean (NCBI access: NP-001237525.1), synthesized by general biosystems (Anhui) Inc. into pET28a vector, transformed into E.coli BL21 (DE 3), after codon optimization, the sucrose synthase was prepared as described in example 2. The sucrose synthase can be replaced in other embodiments with other sucrose synthases of the prior art. Specifically, the reaction mixture (200 mL) contained 50mM potassium phosphate buffer (pH 7.5), 25mM cinnamyl alcohol, 0.5mM UDP, 5% DMSO, 400mM sucrose, 50mg crude enzyme solution of glycosyltransferase or mutant thereof, and 100mg crude enzyme solution of sucrose synthase (excess). The reaction was carried out in a shaker at 30℃and at 200rpm. During the reaction, 50. Mu.L of the reaction product was aspirated at intervals, 150. Mu.L of methanol was added to terminate the reaction, and the progress of the reaction was analyzed by the method described in example 2 after dilution by a certain multiple, and the results are shown in FIG. 6. The results showed that at 24h of reaction, wild-type glycosyltransferase WT-BlYjiC and mutant BlYjiC-F111W and BlYjiC-F175D catalyzed 16.5mM (4.9 g/L), 24.0mM (7.1 g/L) and 23.1mM (6.8 g/L) of the complex plugs, respectively, at 66.0%, 96.0% and 92.4% conversion, respectively, and at 204.2mg/L/h, 295.8mg/L/h, 283.3mg/L/h, respectively, of space-time yield. These results show that the activity of the glycosyltransferase BlYjiC after engineering is significantly improved, and the mutants have great practical application value in the aspect of the synthesis of the helicase.

Example 7 product identification

The product obtained by catalyzing mutant BlYjiC-F111W in example 6 was purified by semi-preparative high performance liquid chromatography and C18 semi-preparative column using 30% methanol as mobile phase. The obtained plug was concentrated by rotary evaporation and freeze-dried, and mass spectrometry was performed, and the results are shown in fig. 7. Subsequently, the obtained powder was dissolved using DMSO-d6 as a solvent and subjected to ¹ H and ¹³ C nuclear magnetic resonance spectroscopy, and the results are shown in FIGS. 8 to 9. The mass spectrum and nuclear magnetic data result proves that the product is a plug.

The present invention has been disclosed in the preferred embodiments, but the invention is not limited thereto, and the technical solutions obtained by adopting equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. An application of glycosyltransferase mutant in catalyzing synthesis of a plug, which is characterized in that:

the glycosyltransferase mutant mutates the 111 th phenylalanine of the amino acid sequence shown in SEQ ID NO.1 into tryptophan; or mutating 175 th phenylalanine of SEQ ID NO.1 into aspartic acid.

2. The application of the genetically engineered bacterium expressing the glycosyltransferase mutant in catalyzing the synthesis of a plug is characterized in that:

The glycosyltransferase mutant mutates the 111 th phenylalanine of the amino acid sequence shown in SEQ ID NO.1 into tryptophan; or mutating phenylalanine at position 175 of SEQ ID NO.1 to aspartic acid; the genetically engineered bacterium is escherichia coli BL21 (DE 3).

3. The use of the glycosyltransferase mutant according to claim 1 in catalyzing the synthesis of a plug or the use of the genetically engineered bacterium expressing the glycosyltransferase mutant according to claim 2 in catalyzing the synthesis of a plug, characterized in that:

under the existence of UDP-glucose provided by a UDP-glucose circulating system, a substrate cinnamyl alcohol is subjected to glycosylation reaction under the catalysis of a glycosyltransferase mutant to generate a plug;

The specific cycle process of the UDP-glucose circulating system is as follows: in the presence of UDP, sucrose is catalyzed by sucrose synthase to generate glucose, fructose and UDP-glucose, and the generated UDP-glucose participates in the synthesis of the plug again;

200 The mL reaction mixture contained 50mM potassium phosphate buffer, 25mM cinnamyl alcohol, 0.5 mM UDP, 5% DMSO, 400 mM sucrose, 50mg glycosyltransferase mutant crude enzyme solution, 100mg sucrose synthase crude enzyme solution, pH of 50mM potassium phosphate buffer 7.5, reaction temperature of 30℃and reaction time of 24h.